Engineering Bacteria to Produce Methyl-Anthranilate, a Grape Flavoring Compound

MANT compound is widely used to impart grape scent and flavor via various applications in food flavoring, cosmetics, perfumes, pharmaceuticals, and agriculture. Owing to the low yields of direct extraction of MANT from natural sources, MANT is currently manufactured by chemical processes, via esterification of petroleum-derived anthranilic acid (ANT) and methanol using homogeneous acids as catalysts. However, these processes suffer from several disadvantages, such as the requirement of large quantities of acid catalysts and problems with the disposal of these toxic liquid acids after use. More importantly, MANT produced by such chemical methods is labeled “artificial flavor”, which does not satisfy the increasing demand by consumers for “naturally or organically” produced flavors.

In an effort to replace the conventional petroleum-based synthesis of MANT, Distinguished Professor Sang Yup Lee and his research team, from the Chemical and Biomolecular Engineering Department of KAIST, exploited a combination of metabolic engineering, synthetic biology, and bioprocess engineering approaches to develop recombinant Escherichia coli and Corynebacterium glutamicum strains capable of producing MANT directly from glucose; process is based on a plant-derived synthetic metabolic pathway. In both engineered microorganisms, MANT production was systematically optimized through the fine-tuning of key enzyme levels, increasing the direct precursor metabolite supply and enhancing the cofactor availability through engineering of the cellular metabolism. Furthermore, in situ two-phase extractive fermentation using tributyrin as an organic extractant was developed both to overcome the cytotoxicity of MANT and ease the downstream purification of MANT. The resulting strains led to the production of 4.47 and 5.74 g/L of MANT, respectively, in minimal media containing glucose as the sole carbon and energy source. This work is the first example of bio-based production of MANT from simple and renewable sugars by microbial fermentation of engineered bacteria.

“This study not only represents a significant breakthrough in the direct fermentative production of the grape favor compound MANT from renewable resources, which had been produced only by petrochemical methods over the past 100 years, but also opens up an avenue toward completely natural, methanol-free, sustainable, consumer- and eco-friendly production of flavor and fragrance compounds used in our daily food and other products.,” said Prof. Sang Yup Lee.

This research was published on May 13, 2019 in the Proceedings of the National Academy of Sciences of the United States of America (PNAS), with the title “Microbial production of methyl anthranilate, a grape flavor compound” (DOI 10.1073/pnas.1903875116).